Prefrontal cortex (PFC) dysfunction is thought to underlie many of the maladaptive behaviors that characterize addiction. Although many studies have investigated how subregions of PFC contribute to cognitive processing and drug seeking, very few studies have examined the functional contribution of individual layers. PFC projection neurons are segregated by their output targets, including segregated sublayers within prelimbic PFC projecting to nucleus accumbens (NAc) core vs. rostromedial tegmental nucleus (RMTg), two structures that often have opposing influences on behavior in rats, including those associated with fear and drug seeking. The current set of studies is aimed at testing the novel hypothesis that projection neuron sublayers are an important organizational feature of PFC, and that sublayer differences in afferents, efferents, and function are critical to the diverse functionality of PFC across a range of behavioral responses. The functional role played by projection neuron sublayers in PFC has been overlooked in addiction studies, and further studies of both the behavioral function and anatomical connectivity are necessary for understanding the role that PFC dysfunction plays in addiction. The first aim of the application will investigate whether PFC projection neuron sublayers projecting to NAc vs. RMTg have independent and dissociable contributions to behavior. These studies will use rodent models of relapse and fear: reinstatement of cocaine seeking in a self-administration paradigm (cue-induced lever pressing), and conditioned fear (cue-induced freezing). c-Fos activation patterns in retrogradely-labeled PFC neurons will be used to determine independent function, and optogenetic inhibition of PFC terminals in NAc vs. RMTg will be used to test opposing functional contributions of these two sublayers. The second aim of the application will use trans-synaptic retrograde tracing technology to investigate differences in afferent connectivity to PFC sublayers projecting to either NAc or RMTg. These basic research studies will demonstrate that projection neuron sublayers in PFC have dissociable roles in addiction, and that discrete segmentation of output projection subpopulations is a key component of PFC organization and function that has been overlooked previously.